Subpopulation Of Pyramidal Neurons Research Articles

Differential distribution of parvalbumin-immunoreactive pericellular clusters of terminal boutons in developing and adult monkey neocortex

Basket cells are GABAergic inhibitory interneurons and known regulators of pyramidal cells, the major class of excitatory neurons in neocortex. Parvalbumin (PV), a calcium binding protein, has been colocalized with GABA in cortical neurons ( Celio, 1986. Science 231: 995–998 ) and has been reported to be present in the terminal boutons of basket neurons forming pericellular clusters in monkey neocortex ( Hendry et al. 1989. Exp. Brain Res. 76: 467–472 ). In this study, we used immunohistochemical methods to evaluate the regional and laminar distributions of PV-immunoreactive (PV-IR) pericellular clusters of terminal boutons in the neocortex of neonatal, infant, adolescent, and adult rhesus monkeys. PV-IR pericellular clusters were composed of labeled terminal boutons that outlined the somata and proximal dendrites of large pyramidal neurons in layers III and V of primary motor cortex, layers V and VI of primary visual cortex, and layer V of visual association cortex (area 18). This laminar pattern was present in neonatal animals and did not change with age in motor cortex. However, in the visual regions of adolescent and adult animals, such PV-IR structures were not detected. PV-positive pericellular clusters were not observed in the prefrontal cortex at any age. The pattern of distribution of PV-containing pericellular clusters paralleled that of a subpopulation of pyramidal neurons containing nonphosphorylated neurofilament proteins (NFP); double labeling studies confirmed that a sub-group of NFP-positive pyramidal neurons were the targets of PV-IR pericellular clusters. The distribution of PV-IR pericellular clusters was compared to that of PV-IR terminal boutons of another class of interneurons, the chandelier cells. Terminal boutons of chandelier neuron axons align in vertical rod-like structures known as cartridges. Subpopulations of chandelier axon cartridges have been previously shown to be PV-IR and their distribution in visual and prefrontal cortices has been described (DeFelipe et al. 1989. Brain Res. 503: 49–54; Lewis and Lund. 1990. J. Comp. Neurol. 293: 599–615). These two types of structures composed of PV-IR terminal boutons tended to be present in different laminae in all regions and ages examined, except in layer III of primary motor cortex where both PV-IR pericellular clusters and chandelier cartridges were found. These findings indicate that in monkey neocortex PV immunoreactivity is present in pericellular clusters of terminal boutons that are likely to arise from basket cells. The differential regional, laminar and developmental patterns of PV immunoreactivity in these structures may reveal their unique pyramidal cell targets and provide insight into their functional roles in monkey neocortex.

Nonphosphorylated neurofilament protein and calbindin immunoreactivity in layer III pyramidal neurons of human neocortex.

Subpopulations of pyramidal neurons in the neocortex have been shown to contain nonphosphorylated neurofilament protein (NPNFP) and calbindin D28K (Morrison et al., 1987; Campbell and Morrison, 1989; Hof et al., 1990; Kobayashi et al., 1990; Hof and Morrison, 1991; Mesulam and Geula, 1991). However, it is not known what relations, if any, exist between the pyramidal neurons containing each of these proteins. In this study, the expression of NPNFP and calbindin immunoreactivity was compared in six regions of human neocortex. Characteristic laminar patterns of immunoreactivity for each protein were seen in most regions examined, and both NPNFP- and calbindin-labeled pyramidal neurons were found in layer III. However, the pyramidal neurons labeled with NPNFP and calbindin differed in several respects. First, the sublaminar distribution of NPNFP-labeled pyramids within layer III differed across regions, ranging from an even distribution throughout the layer in a visual association region (area 18) to a predominance of labeled neurons in the deep half of that layer in a higher association region (area 20). The distribution of calbindin-immunoreactive pyramidal neurons also varied regionally, but in a different manner than that of the NPNFP-labeled neurons. Second, in every region examined, the average size of NPNFP-labeled layer III pyramids was greater than that of calbindin-immunoreactive pyramids. However, there was substantial regional heterogeneity in the extent to which the size distributions of neurons in each of the two populations overlapped. Third, in the regions in which NPNFP- and calbindin-immunoreactive neurons were most similar in size, the amount of colocalization (as identified by double-labeling studies) was also greatest. Similarly, in the regions in which there was minimal overlap in the size of the NPNFP- and the calbindin-immunoreactive neurons, there was minimal colocalization. These regional characteristics of NPNFP- and calbindin-immunoreactive layer III pyramidal neurons have implications for the involvement of these neuronal populations in Alzheimer's disease.

Neocortical neuronal subpopulations labeled by a monoclonal antibody to calbindin exhibit differential vulnerability in Alzheimer's disease

Numerous studies have demonstrated that specific neuronal subtypes display a differential vulnerability to the pathological process in Alzheimer's disease. Large pyramidal neurons are likely to be highly vulnerable, whereas smaller neurons are more resistant to pathology. Using a monoclonal antibody to the calcium-binding protein calbindin, we observed in the human prefrontal cortex distinct populations of labeled cells. First, a subset of heavily stained interneurons was located in layers II and superficial III and in layers V–VI. Second, a subpopulation of pyramidal neurons in the mid and deep parts of layer III displayed a less intense, punctate staining pattern. The interneurons in the superficial layers were unaffected in the Alzheimer's disease cases. Interestingly, in layers V–VI, there was significant cell loss in the interneuron population, but only in the Alzheimer's disease cases with high neurofibrillary tangle densities. The calbindin-immunoreactive pyramidal neurons of layer III were dramatically affected in the disease. Moreover, there was a strong correlation between the extent of the loss of these cells and neurofibrillary tangle counts. These data suggest that calbindin is present in multiple neuronal subpopulations that exhibit a differential vulnerability in Alzheimer's disease and support the hypothesis that the degenerative process involves specific neuronal subsets with particular anatomical and molecular profiles.

A subpopulation of primate corticocortical neurons is distinguished by somatodendritic distribution of neurofilament protein

In recent immunohistochemical studies of human and monkey neocortex we observed that the somatodendritic distribution of neurofilament protein appears to be restricted to a subpopulation of pyramidal neurons. To further characterize this apparent specificity in cytoskeletal organization, combined retrograde tract tracing and immunohistochemical methods were used to examine the extent to which neurons from different cortical areas providing a projection to prefrontal cortex have a somatodendritic distribution of neurofilament proteins. These studies revealed that the proportion of neurons providing a projection from different cortical areas to prefrontal cortex varied from nearly 30% to 90%, and appeared to be related to the functional nature of the projection.

Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer's disease: II. Primary and secondary visual cortex

In this study we investigated the primary and secondary visual areas of normal and Alzheimer's disease brains by using the SMI32 antibody. It is known that in Alzheimer's disease primary sensory areas are usually less devastated than association cortices, although visual symptomatology has been documented early in the course of the disease. In area 17, the SMI32 antibody primarily labeled the perikarya and dentritic tree of the large Meynert cells and cells in layer IVB. Smaller neurons in layers III, V, and VI were also immunoreactive (ir). In area 18, very large SMI32-ir pyramidal neurons in layers III and V were observed. In both areas, staining intensity was correlated with cell size, the largest neurons being the most intensely stained. Only a few changes were observed in the Alzheimer's disease cases. The only statistically significant differences in SMI32-ir neuron counts between control and Alzheimer's disease brains occurred in layer IVB cells and Meynert cells in area 17, and in layer III cells in area 18. In contrast with association cortices, there were no changes in staining intensity in the visual areas. There were fewer neurofibrillary tangles and neuritic plaques in these areas than in prefrontal and inferior temporal cortex, and a correlation between neurofibrillary tangle counts and SMI32-ir neuron loss was only observed in layer III of area 18. These observations show that in the primary and secondary visual cortex, SMI32 also labeled a distinct subset of pyramidal cells that are known from data obtained in the monkey brain to furnish long corticocortical as well as subcortical projections. Interestingly, although there is much less cell and/or neurofibrillary tangle formation in these occipital regions than in prefrontal and temporal association areas, there is significant loss within key subsets of pyramidal cells. The selective loss of this particular subpopulation of pyramidal neurons will disrupt association pathways linking primary visual cortex with areas involved in higher level visual processing. The partial disconnection of such pathways may be relevant to the visual symptomatology frequently observed in Alzheimer's disease patients. These data further support the hypothesis that subtypes of pyramidal neurons with specific anatomical and molecular profiles may display a differential vulnerability in Alzheimer's disease.

Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer's disease: I. Superior frontal and inferior temporal cortex

Various cytoskeletal proteins have been implicated in the cellular pathology of Alzheimer's disease. A monoclonal antibody (SMI32) that recognizes nonphosphorylated epitopes on the medium (168 kDa) and heavy (200 kDa) subunits of neurofilament proteins has been used to label and analyze a specific subpopulation of pyramidal neurons in the prefrontal and inferior temporal cortices of normal and Alzheimer's disease brains. In Alzheimer's disease, the distribution of neuropathological markers predominates in layers III and V in these association areas. In these neocortical regions, SMI32 primarily labels the perikarya and dendrites of large pyramidal neurons, predominantly located within layers III and V. In Alzheimer's disease, a dramatic loss of SMI32-immunoreactive (ir) cells was observed, affecting particularly the largest cells (i.e., cells with a cross-sectional perikaryal area larger than 350 microns 2). The staining intensity of the largest SMI32-ir neurons was significantly reduced in Alzheimer's disease cases, suggesting that an inappropriate phosphorylation of these cytoskeletal proteins may take place in the course of the pathological process. In addition, the SMI32-ir neuron loss and total neuron loss were highly correlated with neurofibrillary tangle counts, whereas such a correlation was not observed with neuritic plaque counts. These quantitative data suggest that SMI32-ir neurons represent a small subset of pyramidal cells that share certain anatomical and molecular characteristics and are highly vulnerable in Alzheimer's disease. Other studies have suggested that SMI32-ir neurons are likely to furnish long corticocortical projections. Thus, their loss would substantially diminish the effectiveness of the distributed processing capacity of the neocortex, resulting in a neocortical isolation syndrome as reflected by the clinical symptomatology observed in these patients. Such correlations between the expression of a selective cellular pathology and specific elements of cortical circuitry will increase our understanding of the molecular and cellular characteristics underlying a given neuronal subclass vulnerability in Alzheimer's disease or other neurodegenerative disorders.

Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca 2+ influx in cultured hippocampal neurons

In several neurological disorders including Alzheimer's disease, abnormal accumulations of cytoskeleton-associated proteins manifest as neurofibrillary tangles (NFTs) in vulnerable brain regions. Antibodies recognizing tau (5E2 and Alz-50) and ubiquitin epitopes in NFTs were used to examine the influence of glutamate and Ca 2+ influx on antigen expression in cultured rat hippocampal neurons. Glutamate caused the degeneration of a subpopulation of pyramidal neurons, which exhibited increased immunostaining with all three antibodies. Subtoxic levels of glutamate also increased 5E2 and Alz-50 antigen levels in a subpopulation of neurons, particularly in the distal regions of the axons. Both glutamate-induced degeneration and increases in tau and ubiquitin immunostaining were prevented by removal of extracellular Ca 2+ Increased immunostaining was also induced by Ca 2+ ionophore A23187 or elevated levels of extracellular K +. The antigenic changes occurred within 1 hr of exposure to glutamate or A23187 and were not prevented by the protein synthesis inhibitor cycloheximide. These data indicate that Ca 2+ influx caused by glutamate can lead to modifications of extant proteins similar to those seen in NFTs.

Development and selective neurodegeneration in cell cultures from different hippocampal regions

Previous studies have shown that pyramidal neurons in hippocampal regions CA1 and CA3 are selectively vulnerable in several neurodegenerative disorders and that a subpopulation of pyramidal neurons in cell cultures of embryonic hippocampus are sensitive to glutamate neurotoxicity. In order to determine whether the patterns of cell loss seen in situ correlate with intrinsic differences in neuronal sensitivities to glutamate-induced denegeration acquired during development, we characterized cultures established from different regions of postnatal rat hippocampus and then examined neuronal sensitivity to glutamate. Tissue corresponding to the dentate gyrus (DG) and regions CA1, CA2, and CA3 of Ammon's horn was removed by microdissection from transverse hippocampal slices and was used to establish cultures of dissociated cells. Cultures from all 4 regions contained 3 major morphological classes of neurons; pyramidal-like, bipolar and stellate. Pyramidal-like neurons comprised the majority of neurons in all cultures; these neurons extended one long and branching axon, and one or more short dendrites. Immunocytochemistry showed that all neurons possessed high levels of glutamate-like and γ-aminobutyric acid (GABA)-like immunoreactivity when grown in isolation. In contrast, when bipolar and pyramidal neurons were cultured in contact with glial cells, glutamate and GABA immunoreactivity were selectively reduced in the bipolar and pyramidal cells, respectively, suggesting that cell interactions influence neurotransmitter phenotype. Subpopulations of hippocampal neurons from each hippocampal region were vulnerable to glutamate-induced neurotoxicity. Bipolar and stellate cells were resistant to glutamate, while pyramidal-like neurons showed varying degrees of sensitivity to glutamate depending upon which region they were taken from. Experiments with specific glutamate receptor agonists and antagonists demonstrated that both non N-methyl- d-spartic acid (NMDA) receptors and NMDA receptors mediated glutamate-induced degeneration. There were clear differences in the vulnerability of the pyramidal-like neuron populations in cultures from the different hippocampal regions. The rank order of the vulnerability of pyramidal-like neurons to glutamate-induced neurodegeneration between regions in culture was: <CA2<CA3<CA1. This pattern of selective vulnerability in cell culture corresponds directly to the pattern of selective cell loss seen in situ in Alzheimer's disease, epilepsy, and stroke suggesting that intrinsic neuronal differences in glutamate sensitivity may be involved in these disorders.

Monoclonal antibody to neurofilament protein (SMI‐32) labels a subpopulation of pyramidal neurons in the human and monkey neocortex

A monoclonal antibody that recognizes a nonphosphorylated epitope on the 168 kDa and 200 kDa subunits of neurofilament proteins has been used in an immunohistochemical study of cynomolgus monkey (Macaca fascicularis) and human neocortex. This antibody, SMI-32, primarily labels the cell body and dendrites of a subset of pyramidal neurons in both species. A greater proportion of neocortical pyramidal neurons were SMI-32 immunoreactive (ir) in the human than in the monkey. SMI-32-ir neurons exhibited consistent differences in the intensity of their immunoreactivity that correlated with cell size. The cellular specificity of SMI-32 immunoreactivity suggests that a subpopulation of neurons can be distinguished on the basis of differences in the molecular characteristics of basic cytoskeletal elements such as neurofilament proteins. The size, density, and laminar distribution of SMI-32-ir neurons differed substantially across neocortical areas within each species and between species. Differences across cortical areas were particularly striking in the monkey. For example, the anterior parainsular cortex had a substantial population of large SMI-32-ir neurons in layer V and a near absence of any immunoreactive neurons in the supragranular layers. This contrasted with the cortical area located more laterally on the superior temporal gyrus, where layers III and V contained substantial populations of large SMI-32-ir neurons. Both areas differed significantly from the posterior inferior temporal gyrus, which was distinguished by a bimodal distribution of large SMI-32-ir neurons in layer III. Differences across human areas were less obvious because of the increase in the number of SMI-32-ir neurons. Perhaps the most notable differences across human areas resulted from shifts in the density of the larger SMI-32-ir neurons in deep layer III. A comparison between the species revealed that isocortical areas exhibited greater differences in their representation of SMI-32-ir neurons than primary sensory or transitional cortical areas. A comparison of distribution patterns of SMI-32-ir neurons across monkey cortical areas and data available on the laminar organization of cortical efferent neurons suggests that a common anatomic characteristic of this chemically identified subpopulation of neurons is that they have a distant axonal projection. Such correlations of cell biological characteristics with specific elements of cortical circuitry will further our understanding of the molecular and cellular properties that are critically linked to a given neuron's role in cortical structure and function.

Roles for mitotic history in the generation and degeneration of hippocampal neuroarchitecture

The mechanisms regulating the highly ordered neuroarchitecture of the mammalian brain are largely unknown. The present study took advantage of hippocampal pyramidal-like neurons that arose from a common progenitor cell in cell culture (sister neurons) to ascertain the contribution of intrinsic factors to both the generation and degeneration of neuroarchitecture. Sister neurons were similar in overall cell form and dendritic numbers and lengths. Control non-sister neurons that grew in contact did not generate similar morphologies, indicating that the similarity of sister cells did not result from influences of the local microenvironment or cell interactions. These results suggest that intrinsic factors related to mitotic history play a role in the generation of neuroarchitecture. Since particular groups of hippocampal neurons are sensitive to glutamate neurotoxicity in situ and are vulnerable in neurodegenerative disorders, it was of interest to test glutamate sensitivity in the neuronal population and in mitotic sister neurons. A subpopulation of pyramidal neurons was sensitive to glutamate neurotoxicity. A striking finding was that sister neurons were invariably either both sensitive or both resistant to glutamate, while non-sister neurons often showed different responses to glutamate. Pharmacological studies indicated that glutamate neurotoxicity was mediated by kainate/quisqualate type receptors by a mechanism involving calcium influx through membrane channels. Fura-2 measurements of intracellular calcium revealed that sister neurons had similar rest levels of calcium and, strikingly, glutamate caused a dramatic increase in intracellular calcium levels only in neurons which subsequently degenerated. Apparently, intrinsic differences in sensitivity to glutamate lie at a point prior to calcium entry, probably at the level of glutamate receptors. Taken together, these results indicate that the mitotic history of a neuron can determine its presence and potential for connectivity as well as its susceptibility to neurodegeneration.

Monoclonal antibody identification of subpopulations of cerebral cortical neurons affected in Alzheimer disease.

Neuronal degeneration is one of the hallmarks of Alzheimer disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, we have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mABs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAB 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1, to the best of our knowledge, do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands on immunoblots of homogenates of normal and AD cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible.

Laminar and regional distributions of neurofibrillary tangles and neuritic plaques in Alzheimer's disease: a quantitative study of visual and auditory cortices

The number of Thioflavine S-positive neurofibrillary tangles (NFT) and neuritic plaques (NP) was determined in visual and auditory cortical regions of 8 patients with Alzheimer's disease. On both a regional and laminar basis, NFT exhibited very distinctive and consistent distribution patterns. The mean (+/- SEM) number of NFT in a 250-micron-wide cortical traverse was very low in area 17, primary visual cortex (0.9 +/- 1.0), increased 20-fold in the immediately adjacent visual association cortex of area 18 (19.7 +/- 3.6), and showed a further doubling in area 20, the higher-order visual association cortex of the inferior temporal gyrus (35.5 +/- 8.8). Similar differences in NFT number were present between primary auditory (1.6 +/- 0.5) and auditory association (18.9 +/- 5.4) regions. On a laminar basis, NFT were predominantly present in layers III and V, although there were striking regional differences in the proportion of NFT in these 2 layers. Layer III contained 79% of the NFT in layers III and V in area 18, 41% in area 20, and only 27% in area 22. In contrast, NP showed different, and less specific, regional and laminar distribution patterns. Total NP number was similar in the 3 visual areas, although there were marked regional differences in the type of NP present. Nearly 80% of the NP in area 17 was of the NPc type (i.e., contained a dense, brightly fluorescent core), whereas over 70% of the NP in both areas 18 and 21 was of the NPnc type (i.e., lacked a dense, brightly fluorescent core). NP were present in every cortical layer but were most numerous in layers III and IV. The distinctive distribution patterns of NFT are very similar to the regional and laminar locations of long corticocortical projection neurons in homologous regions of monkey neocortex. This association suggests that NFT reside in the cell bodies of a subpopulation of pyramidal neurons, namely, those that furnish long corticocortical projections. In contrast, the distribution patterns of NP suggest that multiple neuronal systems contribute to their formation.